The pathogen Leishmania is a protozoan parasite causing serious worldwide morbidity affecting over 200 million individuals. Regulation of the cellular immune response directed against Leishmania is critical for the establishment of effective control of the pathogen, while limiting the development of pathology. Our long-term goal is to identify immunomodulatory leukocyte populations and cytokine networks that are involved in the development of protective or pathogenic immune responses in human leishmaniasis, while at the same time strengthening research capacities in Brazil. We have examined T cell responses in human CL and have determined that a/[unreadable] CD4-CD8-, double negative (DN) T cells, are the second most prevalent cellular source of Th1 type cytokine producing cells. Thus, our aim is to obtain a functional and structural vision of a/[unreadable] DN T cells from patients with CL. Specifically, studying PBMC and/or lesions from CL patients (in aim 4 from mucosal (ML) patients) from an endemic area near Salvador, Brazil, we will determine: 1) What is the relative contribution of a/[unreadable] and ?/d DN T cells to the overall DN T cell profile seen in CL patients? PBMC derived a/[unreadable] vs. ?/d DN T cells will be studied both ex vivo and following antigen stimulation to determine: a) their relative activation state and proliferative capacity (by CFSE staining and flow cytometry), b) their inflammatory and regulatory cytokine production, and c) their cytotoxic capacity (by expression of granzyme or perforin). Multiparameter flow cytometry will be used to address these aims. 2) Do a/[unreadable] DN T cells from CL patients recognize antigens presented via CD1? We will determine: a) which CD1 molecules (CD1a, b, c, or d) are involved in the activation of a/[unreadable] DN T cells (by blocking with mAbs), and b) are a/[unreadable] DN T cells stimulated by CD1/antigen conjugates loaded with artificial (Gal-Cer) or Leishmania derived lipids and/or extracts (using CD1 trasfected cell lines). 3) What is the representation of a/[unreadable] DN T cells in the lesion of CL patients? We will determine: a) the frequency of a/[unreadable] DN T cells in the lesion as compared to the blood, b) the activation state of DN T cells in the lesion as compared to those in the blood, c) the functional profile of DN T cells from the lesion as compared to the blood. We will use flow cytometry and confocal microscopy to address this aim. 4) Does the DN T cell population arise in ML patients? We will determine if mucosal patients express the DN T cell populations in the same frequencies, and with similar or different functional activities to that seen in CL. Public health relevance: Leishmaniasis is a devastating disease that causes suffering and the death of millions worldwide. Thus, determining what factors control the development of protective or pathogenic immune responses could lead to new treatments and vaccines for this parasitic disease.